• Korean Journal of Microbiology
• /
• v.38 no.4
• /
• pp.254-259
• /
• 2002

A psychrotrophic bacterium was isolated from Antarctic marine sediment and identified as Shewanella sp. species based on the biochemical properties and 16S rRNA sequence, and designated as Shewanella sp. L93. Extracellular protease produced by this strain was purified through ammonium sulfate precipitation, High-Q column chromatography, first gel permeation chromatography, BioScale Q2 ion exchange chromatography and second gel permeation chromatography, and basic properties of this enzyme were investigated.

• Macromolecular Research
• /
• v.16 no.1
• /
• pp.1-5
• /
• 2008

The enzymatic hydrolysis of chitin has been studied for almost a century, and early work established that at least two enzymes are required, a chitinase that mainly yields the disaccharide N,N'-diacetylchitobiose, or $(GlcNAc)_2$, and a "chitobiase", or ${\beta}$-N-acetylglucosaminidase, which gives the final product G1cNAc. This pathway has not been completely identified but has remained the central concept for the chitin catabolism through the $20^{th}$ century1 including in marine bacteria. However, the chitin catabolic cascade is quite complex, as described in this review. This report describes three biologically functional genes involved in the chitin catabolic cascade of Vibrios in an attempt to better understand the metabolic pathway of chitin.

• KSBB Journal
• /
• v.14 no.2
• /
• pp.192-197
• /
• 1999

marine bacterium Pseudomonas sp. CHCS-2 produced the biosurfactant in the culture broth which contained 2%(w/v) arabian light crude oil and the productivity of biosurfactant was increased with the addition of glucose. The crude oil in the culture broth was degraded by this strain and carbon chain of $_nC_{12}~_nC_{22}$ was completely degradaded during the incubation for 196 h. The crude biosurfactant was purified by Amberlite XAD-7, Sepharose CL-4B and DEAE-Sepharose CL-6B column chromatography. Therefore, 0.21g/L of the purified biosurfactnat was obtained. The purified biosurfactant was a type of lipoprotein and the molecular weight was estimated as 67kDa by SDS-PAGE. The lipid composition was identified as octadecanoic acid by gas chromatography/mass spectrometry. And then, the N-terminal amino acid sequence of the protein was determined as Ser-Val-lle-Asn-Thr-lle-X-Met-lle-Gly-Gln-Gln- and the sequence did not show homology to any other known lipoprotein. Therefore, the purified lopoprotein was predicted novel biosurfactant.

• KSBB Journal
• /
• v.19 no.4
• /
• pp.295-300
• /
• 2004

A marine bacterium for producing an collagenolytic protease was isolated from the southern sea of Korea and identified as Vibrio vulnificus and named as Vibrio vulnificus CYK279H. This strain producing an collagenolytic protease was showed high activity toward collagen and gelatin as substrate. The optimum initial pH, NaCl, and temperature for cell growth and protease production was 7.5, 2.0% and 25$^{\circ}C$, respectively. Optimization for collagenolytic protease production was composed of 0.3% D-galactose, 0.6% yeast extract, 4.0% gelatin, 0.2% (NH$_4$)$_2$SO$_4$, and 0.2 mM ferric citrate in artificial sea water. The maximum protease production was required gelatin and yeast extract. The collagenolytic protease production by Vibrio vulnificus CYK279H reached a maximum of 73 unit/l after the cultivation for 18 h under the optimized medium.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.3
• /
• pp.476-482
• /
• 2002

Methanol dehydrogenase (MDH) and c-type cytochromes from marine methanol-oxidizing bacterium, Methylophaga sp. MP, were purified and characterized. The native MDH had a molecular mass of 148 kDa and its isoelectric point was 5.5. Two c-type cytochromes, $c_L\;and\;c_H$, were found, and their isoelectric points were 3.4 and 8.0, respectively. The purified MDH had higher thermal stability than that of the other soil methylotrophic bacteria. The electron flow rate from MDH to cytochrome $c_L$was higher than that from MDH to cytochrome $c_H$, indicating that the physiological primary electron acceptor for MDH is cytochrome $c_L$. The electron transfer from MDH to phenazine ethosulfate (PES, artificial electron acceptor) in the two dye (PES/DCPIP)-linked assay system was not inhibited by NaCl, whereas the electron flow from MDH to cytochrome $c_L$ in the cytochrome/DCPIP-linked assay system was suppressed significantly by NaCl. Metal chelating agents such as EDTA showed the same effects on the MDH activity.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.5
• /
• pp.776-783
• /
• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.9
• /
• pp.1237-1244
• /
• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.29 no.5
• /
• pp.637-642
• /
• 1996

We isolated a marine bacterium, Pseudomonas sp,, which could produce the enzyme of alginate lyase, and cloned the alginate lyase gene in Escherichia coli. The cloned DNA was overexpressed with approximately $50\%$ amount of total proteins. In addition, the expressed proteins were not secreted into the medium, and most of them existed in the cytoplasm by the soluble form, but not observed any inclusion body by TIM. For the optimum enzyme activity, temperature was $20^{\circ}C$, pH was 7.0, and Km and Vmax values of the enzyme were $0.4\%$ and 625 units/mg, respectively.

• KSBB Journal
• /
• v.31 no.2
• /
• pp.113-119
• /
• 2016

A novel agar-degrading marine bacterium, SH-1 strain, was isolated from seashore of Namhae at Gyeongnam province, Korea. The SH-1 strain exhibited 98% similarity with Alteromonas species based on 16S rDNA sequencing and named as Alteromonas sp. SH-1. Alteromonas sp. SH-1 showed agarase activity of 348.3 U/L (1.67 U/mg protein). The molecular masses of the enzymes were predicted as about 85 kDa and 110 kDa by SDS-PAGE and zymogram. The enzymatic activity was optimal at $30^{\circ}C$ and the relative agarase activity was decreased as temperature increase from $30^{\circ}C$ and thus about 90% and 70% activities were shown at $40^{\circ}C$ and $50^{\circ}C$, respectively. The optimum pH was 6.0 for agarase activity in 20 mM Tris-HCl buffer and activities were less than 70% and 85% activity at pH 5.0 and pH 7.0, respectively, compared with that at pH 6. Agarase activity has remained over 90% at $20^{\circ}C$ after 1.5 hour exposure at this temperature. However, its activity was less than 60% at $30^{\circ}C$ after 0.5 h exposure at this temperature. The enzymes produced agarooligosaccharides such as agaropentaose and agarotriose from agarose, indicating that the agarases are ${\alpha}$-agarases. Thus, Alteromonas sp. SH-1 and its agarases would be useful for the industrial production of agarooligosaccharides which are known as having anticancer and antioxidation activities.

• Microbiology and Biotechnology Letters
• /
• v.41 no.1
• /
• pp.8-16
• /
• 2013

An agar-hydrolyzing marine bacterium, strain GNUM08120, was isolated from Sargassum fulvellum collected from Yeongil bay of East Sea of Korea. The isolate was Gram-negative, aerobic, motile with single polar flagellum, and grew at 1-10% NaCl, pH 5.0-8.0, and $15-37^{\circ}C$. G+C content and the predominant respiratory quinone were 46.13 mol% and Q-8, respectively. The major cellular fatty acids were Summed feature 3 (24.5%), $C_{16:0}$ (21.7%), and $C_{18:1}{\omega}7c$ (12.5%). Based on 16S rRNA gene sequence similarity and DNA-DNA hybridization analyses, strain GNUM08120 was identified as a novel subspecies of Alteromonas macleodii, designated Alteromonas macleodii subsp. GNUM08120. Production of agarase by strain GNUM08120 was likely repressed by the effect of carbon catabolite repression caused by glucose. The crude agarase prepared from 12-h culture broth of strain GNUM08120 exhibited an optimum pH and temperature for agarase activity at 7.0 and $40^{\circ}C$, respectively. The crude enzyme produced (neo)agarobiose, (neo)agarotetraose, and (neo)agarohexaose as the hydrolyzed product of agarose.